(a) Field of the Invention
The invention relates to a method of designing agonistic and/or antagonistic antibodies for any hormone receptor more specifically antibodies which are capable of blocking nerve growth factor (NGF) binding and antibodies which can mimic NGF binding to its receptor.
(b) Description of Prior Art
The TrkA receptor is a 140 kDa transmembrane glycoprotein with tyrosine kinase activity that functions as the Nerve Growth Factor (NGF) receptor. NGF also binds with low affinity to a p75 receptor whose signaling function is unclear. Homo or heterodimers or oligomers of TrkA and p75 bind NGF with higher affinity (Jing, S. et al. (1992) Neuron, 9: 1067-1079) suggesting that specific receptor conformations may play specific functions.
NGF promotes the differentiation of certain neuronal cells, is mitogenic for TrkA-transfected fibroblasts, and allows survival in serum-deprived conditions for both cell types. Activation of the tyrosine kinase activity of TrkA via UGF binding leads to receptor trans- and auto-tyrosine phosphorylation (PY), and PY of second messengers including phosphatidylinositol-3 kinase (PI-3 kinase). PI-3 kinase is involved in protein trafficking and endocytosis of ligand-receptor complexes (reviewed by Kaplan, D. R. et al. (1994) J. Neurobiol., 25: 1404-1417). Since microinjection of NGF into cells does not result in NGF biological signals, cell surface receptor ligation and internalization of TrkA or NGF-TrkA complexes must mediate these effects.
TrkA, like most kinase growth factor receptors, signals through receptor oligomerization. Thus, mono-valent TrkA-binding agents are antagonistic or have no biological effects (LeSauteur, L. et al.(1995) J. Biol. Chem., 270:6564-6569), whereas bivalent receptor-binding agents such as NGF (a homodimer) or antibodies can be agonistic. The principle of using polyclonal antibodies to activate neural receptors has been demonstrated (Twyman, R. E. et al. (1995) Neuron, 14: 755-762). In contrast, only a limited number of anti-receptor monoclonal antibodies (mAb) mimic ligand functions (Taub, R. et al. (1992) Biochemistry, 31: 7431-7435), and none exist against neurotrophin receptors.
It would be highly desirable to be provided with an agonistic or antagonistic anti-human TrkA mAb which recognizes the NGF docking site. Such antibodies may be used for the diagnosis, treatment or prevention of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors. Also these antibodies may be used to develop and screen for pharmaceutical agents which are agonistic or antagonistic by binding to the TrkA receptors.
One aim of the present invention is to report the development and characterization of an agonistic anti-human TrkA mAb 5C3 which recognizes at least one NGF docking site. This MAb 5C3 was used to characterize the pattern of TrkA protein expression in normal human brain, and the NGF binding features of the receptor. MAb 5C3 behaves like NGF in bioassays, and monomeric 5C3 Fabs retained binding and functional agonistic activity. MAb 5C3 will be useful to identify the NGF docking site on TrkA and possibly as a pharmacological lead in the development of small mimetics.
In accordance with the present invention there is provided an antibody or functional fragment thereof which binds to at least the TrkA receptor under physiological conditions, and wherein the binding to the receptor at least partially mimics or inhibits nerve growth factor biological activity.
In accordance with the present invention there is also provided a method of screening pharmacological agents which are capable to mimic or inhibit nerve growth factor biological activity, which comprises using the antibody of the present invention to screen for pharmacological agents capable of binding to the complementary determining region of the antibody, wherein the screened pharmacological agents can mimic or inhibit nerve growth factor biological activity.
In accordance with the present invention there is also provided the use of pharmacological agents obtained by the process of the present invention, for the in vivo inhibition of nerve growth factor binding to TrkA receptor or the internalization or downmodulation of the receptor.
In accordance with the present invention there is also provided the use of the antibody of the present invention, for the in vivo inhibition of nerve growth factor binding to TrkA receptor or the internalization or downmodulation of the receptor, such as for inhibiting tumor growth in situ, for the treatment or prevention of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors, for mapping hormone-receptor interactive sites and receptor domain-function correlation such as mapping TrkA docking sites, for screening pharmacological agents which bind to the hormone-receptor interactive sites.
In accordance with the present invention there is also provided an antibody having CDR-like domains of hormones, wherein the antibody or functional fragment thereof binds to at least TrkA receptor under physiological conditions, and wherein the binding to the receptor at least partially mimics or inhibits nerve growth factor biological activity.
In accordance with the present invention there is also provided a method for the treatment of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors in a patient, which comprises administering an effective amount of an antibody of the present invention or a functional fragment thereof to a patient.
In accordance with the present invention there is also provided a pharmaceutical composition for the treatment of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors, which comprises an effective amount of an antibody of the present invention or a functional fragment thereof in association with a pharmaceutically acceptable carrier.
In accordance with the present invention there is also provided a method for immunization of a mammal against an antibody of the present invention or a functional fragment thereof, which comprises administering by systemic injection an immunizing amount of at least one of the antibody or the fragment thereof in an immunogenic form in association with a pharmaceutically acceptable carrier.
In accordance with the present invention there is also provided a method for the prognosis or diagnosis of human tumors which comprises:
a) biopsy and immunocytochemistry of tumors using the antibody of the present invention and fragments thereof; or
b) radiolabeling of the antibody of the present invention and fragments thereof and nuclear imaging analysis.
In accordance with the present invention there is also provided a method for the treatment of human tumor of a patient which comprises the steps of:
a) coupling cytotoxic agents to the antibody of the present invention and fragments thereof;
b) administering the coupled antibody of step a) to the patient.
In accordance with the present invention there is also provided a pharmaceutical composition for the targeting of pharmaceutical agents to tissues of the central and/or peripheral nervous system, which comprises an effective amount of an antibody of the present invention or a functional fragment thereof coupled to a pharmaceutical agent in association with a pharmaceutically acceptable carrier. The pharmaceutical agent may be selected from the group consisting of radioligands, nucleic acid molecules, toxins, growth factors and gangliosides.
In accordance with the present invention there is also provided a TrkA docking site which binds to the antibody of the present invention.
In accordance with the present invention there is also provided the use of the docking site of the present invention for screening pharmacological agents which are capable to mimic or inhibit nerve growth factor biological activity and said antibody biological antibody.